ARTICLE IN PRESS

International Dairy Journal 15 (2005) 989–997

www.elsevier.com/locate/idairyj

Effect of temperature on growth and metabolism of probiotic

bacteria in milk
Hilde M. Østlie, Janneke Treimo, Judith A. Narvhus
Department of Chemistry, Biotechnology and Food Science, Agricultural University of Norway, P. Box 5003, N-1432 Ås, Norway
Received 24 February 2003; accepted 25 August 2004

Abstract

The growth and metabolism of six probiotic strains with documented health effects were studied in ultra-high temperature (UHT)
treated milk supplemented with 0.5% (w/v) tryptone or 0.75% (w/v) fructose at different temperatures. The probiotic strains were
Lactobacillus acidophilus La5, Lb. acidophilus 1748, Lb. johnsonii LA1, Lb. rhamnosus GG, Lb. reuteri SD 2112 and Bifidobacterium
animalis BB12. Fermentation was followed for 48 h at 20, 30, 37 and 45 1C and the samples were analysed for pH, log cfu mL 1,
volatile compounds, organic acids and carbon dioxide. All six probiotic strains showed very different profiles of metabolites during
fermentation, however, the two Lb. acidophilus strains were the most alike. All strains, except Lb. reuteri SD 2112, showed viable cell
numbers above 6.5 log cfu mL 1 after 48 h fermentation at 30, 37 and 45 1C. The probiotic strains produced different amounts of
metabolic products according to temperature and fermentation time illustrating the importance of controlling these parameters.
r 2004 Elsevier Ltd. All rights reserved.

Keywords: Fermented milk; Probiotic bacteria; Organic acids; Volatile compounds; CO2

1. Introduction Saarela, Mogensen, Fondén, Mättö, and Mattila–Sand-

During the past twenty years there has been a
tremendous increase in the worldwide sales of cultured
products containing probiotic bacteria. Today, most
probiotic strains are used in yoghurts, fermented milks,
ice creams and pharmaceutical products for their
anecdotal health effect (Mattila–Sandholm, 1999).
Increasing knowledge underlines the important role of
the intestinal flora for maintaining health and in the
prevention of disease. Probiotics offer dietary means to
support the balance of intestinal flora (Holzapfel,
Haberer, Snel, Schillinger, & Huis in’t Veld, 1998).
The microorganisms primarily associated with this
balance are lactobacilli and bifidobacteria.
Several aspects have to be taken into consideration in
the selection process of probiotic organisms. Safety
aspects reviewed lately by Salminen et al. (1998) and
Corresponding author. Tel.: +47 64 94 85 78; fax: +47 64 94 37 89.
E-mail address: hilde.ostlie@ikbm.nlh.no (H.M. Østlie).
holm (2000) include specifications as to human origin,
non-pathogenicity and antibiotic resistance character-
istics. Factors related to the technological and sensory
aspects of the probiotic food products are of utmost
importance since only by satisfying the demands of
consumers can the food industry succeed in promoting
the consumption of functional products in the future
(Mattila–Sandholm, Myllärinen, Crittenden, Mogensen,
Fondén, & Saarela, 2002). To maintain confidence in
probiotic products it is important to demonstrate good
survival of the bacteria in food products during their
specified shelf life. In order for any beneficial effect in
humans to develop, the viable cell count should be
above 6 log cfu g 1 in order to supply a sufficient ‘‘daily
dose’’ of 106–109 viable bacteria (Samona & Robinson,
1991; Lee & Salminen, 1995; Vinderola, Bailo, &
Reinheimer, 2000). In addition, a pleasant taste and an
attractive texture are essential for all food products,
regardless of the ‘‘health message’’ of the product
(Saxelin, Grenov, Svensson, Fondén, Reniero, &

0958-6946/$ - see front matter r 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.idairyj.2004.08.015
 ARTICLE IN PRESS
990 H.M. Østlie et al. / International Dairy Journal 15 (2005) 989–997
Mattila-Sandholm, 1999). The final concentration of
lactate should be about 8000 mg kg 1 and the pH
between 4.2 and 4.4 in order for the sensoric qualities
of sourness and firm coagulum to be satisfactory
(Narvhus, 1996).
There is limited published information concerning the
technological production of fermented probiotic milk
products and of the metabolic pathways followed by
specific probiotic organisms during the fermentation of
milk. The shift in metabolic pathways in response to
environmental conditions is well documented in the
literature in the case of homofermentative and hetero-
fermentative lactobacilli (Axelsson, 1998). However, no
information is available about the shift in metabolism of
probiotic bacteria grown in milk in response to
environmental changes. Metabolic changes are very
important from a technological standpoint, since the
amount of organic acids and volatile compounds is
important in the development of flavour and texture of
the fermented product. However, the metabolic changes
may also be important for the microorganisms to obtain
energy and to maintain the NAD+/NADH+H+
balance (Axelsson, 1998; Lopez de Felipe & Hugenholtz,
1999).
Production of fermented milk products using probio-
tic lactic acid bacteria is a major challenge to dairies as
milk is not, on the whole, a good growth medium for
these organisms. Growth and metabolism of five
probiotic strains in ultra-high temperature (UHT) milk
supplemented with tryptone and fructose at 37 1C have
recently been studied by Østlie, Helland, and Narvhus
(2003). In this study, the effect of different incubation
temperatures on the growth and metabolism of six
probiotic strains with documented health effects were
studied in ultra-high temperature (UHT) semi-skimmed
milk supplemented with 0.5% tryptone (w/v) or 0.75%
fructose (w/v). Their ability to produce organic acids,
volatile compounds and carbon dioxide at different
temperatures was focused on.
2. Materials and methods
2.1. Bacterial strains and culture conditions
Lactobacillus johnsonii LA1 was kindly supplied from
Nestec Ltd, Lausanne, Switzerland; Lb. rhamnosus GG
(ATCC 53103) from Valio Ltd, Helsinki, Finland; Lb.
reuteri SD 2112 from Biogaia Biologics, Stockholm,
Sweden; and Lb. acidophilus LA-5 and Bifidobacterium
animalis BB12 from Christian Hansen, Oslo, Norway.
Lb. acidophilus NCFB 1748 was obtained from the
National Collection of Food Bacteria, Reading, Eng-
land. The strains were subcultured three times in de Man
Rogosa Sharpe (MRS) broth (Difco Labs., Detroit, MI,
USA) at 37 1C overnight, before a final inoculation for
making concentrated stock cultures. Cysteine hydro-
chloride (0.05%, w/v, Sigma, St. Louis, MO, USA) was
added to MRS broth for culturing B. animalis BB12 and
Lb. johnsonii LA1. Frozen concentrated stock cultures
were made as described by Østlie et al. (2003).
The concentrated cultures (10  ) were stored in 3 mL
lots at 80 1C.
2.2. Production of fermented milk
One bottle containing 300 mL UHT milk (1.5% (v/v)
fat, TINE, Oslo, Norway) was inoculated with 1% (v/v)
of the frozen culture. One bottle was inoculated for each
incubation temperature. The milk to be inoculated with
Lb. rhamnosus GG was supplemented in advance with
0.75% (w/v) filter-sterilized fructose (Merck, Darm-
stadt, Germany) and the milk for the other strains was
supplemented with 0.5% (w/v) filter sterilized tryptone
(Oxoid Ltd., Hampshire, England). Forty millilitre lots
of the inoculated milk were aseptically distributed in
50 mL sterile bottles. One bottle was prepared for each
sampling time and was used for all analyses except the
CO2 measurement. For CO2 measurement, 10 mL lots of
the freshly inoculated milk were aseptically distributed
into sterile headspace vials (20-CV, Chromacol Ltd,
Trumbell, USA) and sealed with sterile septa (20-CB3,
Chromacol Ltd) and aluminium crimp caps (20-ACB3,
Chromacol Ltd). One vial was prepared for each
sampling time. Both bottles and vials were incubated
at 20, 30, 37 and 45 1C for 0–48 h. Viable microbial
counts, pH, volatile compounds, organic acids and
carbon dioxide were determined in the incubated milk
after 0, 4, 8, 12, 18, 24 and 48 h incubation.
2.2.1. Viable microorganisms
Samples were diluted in peptone–saline water (0.9%,
w/v saline; 0.1%, w/v peptone) and viable counts of the
probiotic strains were determined on MRS agar (Difco)
after anaerobic incubation at 37 1C for 3 days (BBL
GasPakPlus System, Becton Dickinson Microbiology
Systems, Sparks, MD, USA).
2.2.2. Chemical analysis
All pH measurements were made during fermentation
using a Radiometer (pHM 92) pH meter with a
combined glass electrode and temperature probe
(Radiometer, Copenhagen, Denmark). The pH meter
was calibrated using standard buffer solutions (Merck)
at pH 4.0 and 7.0.
Volatile compounds were analysed by headspace gas
chromatography according to the method of Narvhus,
Østeraas, Mutukumira, and Abrahamsen (1998) as
described by Østlie et al. (2003).
Organic acids were analysed by high pressure liquid
chromatography (HPLC) using a modification of the
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H.M. Østlie et al. / International Dairy Journal 15 (2005) 989–997 991

method of Marsili, Ostapenko, Simmons, and Green

(1981) as described by Østlie et al. (2003).
Carbon dioxide production was determined by an
infra red (I.R.) gas analyser (ADC 225 Mk3, Analytical
development Co. Ltd., Hoddesdon, Hertfordshire, UK)
by the method of Narvhus, Hulbækdal, Baugerød, and
Abrahamsen (1991) as described by Østlie et al. (2003).

3. Results and discussion

Results presented are based on growth and metabo-

lism experiments at different temperatures. Figures
represent the average value of three replicates. No
growth or metabolism was seen at 20 1C for Lb.
acidophilus La5, Lb. acidophilus 1748, Lb. reuteri SD
2112 and B. animalis BB12, however, Lb. rhamnosus GG
and Lb. johnsonii LA1 showed some growth and
metabolism at this temperature and the main findings
will be presented.

3.1. Viable microorganisms and pH

Growth of the strains in UHT milk varied consider-

ably according to incubation temperature (Fig. 1). All
strains except B. animalis showed the most rapid
increase and the highest viable cell number in milk
incubated at 37 1C, attaining viable cell numbers of
8.65–9.21 log cfu mL 1 after 12–24 h incubation. About
the same maximum log cfu mL 1 was seen after different
incubation times at the other temperatures. Lb. reuteri
SD 2112 showed the fastest growth of all strains,
showing maximum viable cells after 8 h incubation at
37 1C and after only 4 h incubation at 45 1C. B. animalis
BB12 grew slowly at 30 1C, but attained the highest
increase (1.5 log unit) and the highest viable cell number
(9.6 log cfu mL 1) of all strains after 48 h incubation at
this temperature. Baron, Roy, and Vuillemard (2000)
studied bifidobacteria and found that fermented milk
produced at 30 1C by different species of bifidobacteria
showed no or only slight increase of viable cells for all
tested species of bifidobacteria. On the other hand,
during fermented milk production at 35 1C the number
of viable cells increased by approximately one log unit
for B. breve, B. longum and B. bifidum whereas there was
no growth observed for the B. infantis strains (Baron et
al., 2000). The stability of the number of viable cells was
best at 30 and 37 1C in our study. After incubation for
24–48 h at 45 1C a reduction in viable cells of
0.93–1.27 log cfu mL 1 was observed for Lb. acidophilus
La5 and 1748 and Lb. rhamnosus GG. Lb. reuteri SD
2112 decreased drastically after only 12 h incubation at
45 1C. However, after 48 h incubation, the viable cell
count of all strains incubated at 30, 37 and 45 1C was
still above 6.5 log cfu mL 1, except for Lb. reuteri SD
2112 incubated at 45 1C for 24 h or more.
Fig. 1. Extent of growth during fermentation at (a) 30 1C, (b) 37 1C
and (c) 45 1C with B. animalis BB12 (~), Lb. acidophilus La5 (&), Lb.
acidophilus 1748 (K), Lb. reuteri SD2112 (m) and Lb. johnsonii LA1
(W) in UHT milk supplemented with 0.5% (w/v) tryptone and with
Lb. rhamnosus GG (’) in UHT milk supplemented with 0.75% (w/v)
fructose. Vertical lines represent standard deviations.
Depending on the probiotic strain, pH decreased from
6.7 to 4.1–5.1, 3.8–4.7 and 3.8–4.5 after 48 h incubation
at 30, 37 and 45 1C, respectively (Fig. 2). Samples
fermented at 45 1C showed a faster reduction in pH early
in the incubation period compared with samples
incubated at 37 1C, except B. animalis BB12. However,
after 12–48 h incubation the pH of the samples
incubated at 37 and 45 1C showed about the same final
pH. This observation agrees with Narvhus et al. (1998),
who studied production of fermented milk at 22, 30 and
37 1C by lactococci and observed that products incu-
bated at 37 1C showed a faster reduction in pH early in
the fermentation period but that after 18 h the products
incubated at 30 and 37 1C showed the same final pH. All
the probiotic strains caused a further decrease in pH on
prolonged incubation between 24 and 48 h at 30 1C.
However, after 24 h incubation at 37 1C, Lb. reuteri SD
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992 H.M. Østlie et al. / International Dairy Journal 15 (2005) 989–997

reaches 4.4 (pH external 3.5). Under starvation condi-

tions in acidic media a race would take place between
the H+ efflux driven by the diminished supply of energy
substrate and the inward leak of H+ facilitated by the
end-products. When the cytoplasmic pH reaches a pH
lower than the allowable threshold pH, the cell dies
(Kashket, 1987).

3.2. Organic acids, volatile organic compounds and

carbon dioxide

The amount of lactic acid produced varied according

to strain and temperature and was generally highest at
37 1C (Fig. 3). The homofermentative Lb. acidophilus
La5 and Lb. acidophilus 1748, and the heterofermenta-
tive Lb. reuteri SD 2112 produced the greatest amounts
at all temperatures. Lb. rhamnosus GG ceased lactic acid

Fig. 2. Changes in pH during fermentation at (a) 30 1C, (b) 37 1C and

(c) 45 1C with B. animalis BB12 (~), Lb. acidophilus La5 (&), Lb.
acidophilus 1748 (K), Lb. reuteri SD2112 (m) and Lb. johnsonii LA1
(W) in UHT milk supplemented with 0.5% (w/v) tryptone and with
Lb. rhamnosus GG (’) in UHT milk supplemented with 0.75% (w/v)
fructose. Vertical lines represent standard deviations.

2112, B. animalis BB12 and Lb. rhamnosus GG showed a

stable pH during prolonged incubation from 24 to 48 h
and Lb. acidophilus 1748, Lb. acidophilus La5 and Lb.
Johnsonii LA1 showed a further decrease in pH. After
48 h incubation at 20 1C Lactobacillus johnsonii LA1
showed no pH reduction in milk but a decrease in viable
cell numbers from 7.4 to 6.6 log cfu mL 1 and Lb.
rhamnosus GG showed a decrease in pH to 5.1 and no
change in viable cell numbers (results not shown).
After incubation of Lb. reuteri SD 2112, B. animalis
BB12 and Lb. johnsonii LA1 for more than 12 h at 45 1C
and of Lb. rhamnosus GG, Lb. acidophilus La5 and 1748
for more than 24 h at 45 1C, all strains showed a marked Fig. 3. Production of lactic acid during fermentation at (a) 30 1C, (b)
37 1C and (c) 45 1C with B. animalis BB12 (~), Lb. acidophilus La5
reduction in viable cells. At this point the pH varied
(&), Lb. acidophilus 1748 (K), Lb. reuteri SD2112 (m) and Lb.
from 3.9 to 4.1 for all strains except B. animalis BB12 johnsonii LA1 (W) in UHT milk supplemented with 0.5% (w/v)
(pH 5.4) and Lb. johnsonii LA1 (pH 4.9). Kashket (1987) tryptone and with Lb. rhamnosus GG (’) in UHT milk supplemented
reported that lactobacilli grow until the cytoplasmic pH with 0.75% (w/v) fructose. Vertical lines represent standard deviations.
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production completely after 24 h incubation at 37 and

45 1C and Lb. reuteri SD 2112 after 12 h incubation at
45 1C, whereas the other four strains continued produ-
cing lactic acid during further incubation at the different
temperatures.
Acetic acid levels were highest in milk inoculated with
B. animalis BB12 at all temperatures, ranging from 2929
to 6901 mg kg 1 after 24–48 h incubation (results not
shown). The other strains produced 173–1171 mg kg 1
after incubation at 30, 37 and 45 1C, where Lb.
rhamnosus GG produced the lowest amounts and Lb.
acidophilus 1748 and Lb. reuteri SD 2112 the highest
amounts at all incubation temperatures. All strains
showed the highest acetic acid levels after incubation at
45 1C, except B. animalis BB12 and Lb. reuteri SD 2112,
that produced the highest amounts after incubation at
37 1C. Lb. johnsonii LA1 and Lb. rhamnosus GG did not
produce acetic acid at 20 1C.
The bifidus pathway used by B. animalis BB12 should
theoretically yield acetate and lactate in a molar ratio of
3:2 (Scardovi, 1986). However, in this study, incubation
temperature greatly influenced this ratio. After incuba-
tion at 30 and 37 1C for 48 h the measured acetate to
lactate ratio was 3:2, but after incubation at 45 1C the
ratio was 1.7:2. Organoleptically, it is important that the
level of acetic acid not is too high in fermented products.
The initial level of citric acid in the milk was 1, 8–2,
1 g kg 1. Lb. reuteri SD 2112, Lb. acidophilus La5 and
Lb. acidophilus 1748 were able to metabolise citrate at all
temperatures, but the rate of reduction varied consider-
ably according to strain and incubation temperature
(Fig. 4). These strains showed an increase in the rate of
reduction with increasing incubation temperature. Inter-
estingly, B. animalis BB12 showed no metabolism of
citrate at 30 and 37 1C, whereas nearly all the citrate was Fig. 4. Development of citric acid during fermentation at (a) 30 1C, (b)
metabolized between 24 and 48 h incubation at 45 1C. 37 1C and (c) 45 1C with B. animalis BB12 (~), Lb. acidophilus La5
(&), Lb. acidophilus 1748 (K), Lb. reuteri SD2112 (m) and Lb.
The concentrations of pyruvate, orotic acid, succinic
johnsonii LA1 (W) in UHT milk supplemented with 0.5% (w/v)
acid and uric acid were also monitored during fermenta- tryptone and with Lb. rhamnosus GG (’) in UHT milk supplemented
tion (results not shown). Pyruvate increased during early with 0.75% (w/v) fructose. Vertical lines represent standard deviations.
fermentation (0–24 h) for most of the strains, but
decreased during prolonged incubation. Lb. acidophilus
1748 produced the peak amount of pyruvate, 85.5, 101 johnsonii LA1 at 45 1C. However, after incubation at
and 135 mg kg 1 after incubation for 12 h at 45 1C, 24 h 30 1C most of the strains showed a reduction in the level
at 37 1C and 48 h at 30 1C, respectively. The other strains of succinic acid, except Lb. rhamnosus GG and Lb.
produced 4.8–31.3 mg kg 1 and these maximum values reuteri SD 2112 which showed an increase in the level of
were achieved between 4 and 18 h incubation. Lb. succinic acid from 880 mg kg 1 to 1548–1726 mg kg 1.
johnsonii LA1 did not produce pyruvate at any The level of uric acid was stable at approximately
temperature. A reduction in the level of orotic acid 15 mg kg 1 during the 48 h fermentation at 30, 37 and
from approximately 72 mg kg 1 to 17.8–68 mg kg 1 after 45 1C for all strains.
incubation at 30, 37 and 45 1C was observed for all The amount of acetaldehyde produced varied greatly
strains. At all temperatures, the greatest reduction in both among the organisms tested and according to
orotic acid was seen for Lb. rhamnosus GG and the least different incubation temperatures. Lb. acidophilus La5,
for Lb. reuteri SD 2112. Succinic acid increased from an Lb. acidophilus 1748, Lb. johnsonii LA1 and B. animalis
initial 880 mg kg 1 to 931–1960 mg kg 1 during the 48 h BB12 produced acetaldehyde (Fig. 5) at all incubation
fermentation at 37 and 45 1C for all strains, except Lb. temperatures, except Lb. johnsonii LA1 at 20 1C, and Lb.
johnsonii LA1 and B. animalis BB12 at 37 1C and Lb. reuteri SD2112 and Lb. rhamnosus GG did not. The
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994 H.M. Østlie et al. / International Dairy Journal 15 (2005) 989–997

among dairy products. In cheese, cultured buttermilk

and butter, acetaldehyde is not desirable but in yoghurt
from 10 to 40 mg kg 1 are required to give the
characteristic flavour (Tamime & Robinson, 1999).
Gonzalez et al. (1994) studied acetaldehyde production
by probiotic bacteria in fermented milk and found that
Lb. acidophilus can form acetaldehyde from different
sources such as carbohydrates, amino acids (such as
threonine) and nucleic acids.
Heterofermentative Lb. reuteri SD2112 produced
considerably higher amounts of ethanol than the other
strains at all temperatures (Fig. 6). After 48 h incuba-
tion, this strain produced 4831–6448 mg kg 1 at
30–45 1C. B. animalis BB12 produced 95 mg kg 1
ethanol after 48 h incubation at 45 1C but only
8.2 mg kg 1 ethanol after 48 h incubation at 30 1C. The
other strains produced 1.3–13 mg kg 1 ethanol after 48 h
at 30–45 1C. Lb. johnsonii LA1 and Lb. rhamnosus GG
produced 13.7 and 6.4 mg kg 1 after incubation at 20 1C.

Fig. 5. Production of acetaldehyde during fermentation at (a) 30 1C,

(b) 37 1C and (c) 45 1C with B. animalis BB12 (~), Lb. acidophilus La5
(&), Lb. acidophilus 1748 (K), Lb. reuteri SD2112 (m) and Lb.
johnsonii LA1 (W) in UHT milk supplemented with 0.5% (w/v)
tryptone and with Lb. rhamnosus GG (’) in UHT milk supplemented
with 0.75% (w/v) fructose. Vertical lines represent standard deviations.

highest amounts of acetaldehyde were produced by Lb.

acidophilus La5 and Lb. acidophilus 1748 after 48 h
incubation at 45 1C: 91.1 and 109.9 mg kg 1, respec-
tively. These high levels would probably not be
organoleptically acceptable and these results therefore
indicate that the acetaldehyde production by these
strains must be limited by controlling the fermentation
time and/or temperature. Lactic acid bacteria form
varying amounts of ethanol and acetaldehyde (Gonza-
lez, Morata de Ambrosini, Manca de Nadra, Pesce de
Ruiz Holgado, & Oliver, 1994). The accumulation of
acetaldehyde in the growth medium can occur when the Fig. 6. Production of ethanol during fermentation at (a) 30 1C, (b)
37 1C and (c) 45 1C with B. animalis BB12 (~), Lb. acidophilus La5
specific activities of enzymes that form acetaldehyde are
(&), Lb. acidophilus 1748 (K), Lb. reuteri SD2112 (m) and Lb.
higher than those able to convert it to ethanol (Gonzalez johnsonii LA1 (W) in UHT milk supplemented with 0.5% (w/v)
et al., 1994). The amount of acetaldehyde required for tryptone and with Lb. rhamnosus GG (’) in UHT milk supplemented
the development of a characteristic flavour varies widely with 0.75% (w/v) fructose. Vertical lines represent standard deviations.
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H.M. Østlie et al. / International Dairy Journal 15 (2005) 989–997
Milk contains an average of 0.2% citrate which is
cometabolised with sugars by many lactic acid bacteria
(Cocaign-Bousquet, Garrigues, Loubiere, & Lindley,
1996). The ability to metabolize citrate to CO2, acetate,
diacetyl, acetoin and sometimes 2, 3 butandiol is a
common trait of many lactic acid bacteria, specially
Lactococcus and Leuconostoc spp. (Hugenholtz, 1993).
All the lactic acid bacteria that can produce diacetyl are
also able to reduce it to butandiol (Hugenholtz, 1993).
Although Lb. acidophilus La5 and Lb. acidophilus 1748
metabolized citrate, no diacetyl and acetoin (Fig. 7) was
Fig. 7. Production of acetoin during fermentation at (a) 30 1C, (b)
37 1C and (c) 45 1C with B. animalis BB12 (~), Lb. acidophilus La5
(&), Lb. acidophilus 1748 (K), Lb. reuteri SD2112 (m) and Lb.
johnsonii LA1 (W) in UHT milk supplemented with 0.5% (w/v)
tryptone and with Lb. rhamnosus GG (’) in UHT milk supplemented
with 0.75% (w/v) fructose. Vertical lines represent standard deviations.
995
detected. It is possible that diacetyl and acetoin were
further metabolized to 2, 3 butandiol, which was not
measured in this study. Lb. reuteri SD 2112 metabolized
citrate and low amounts of diacetyl (0.6–1.6 mg kg 1)
and acetoin (1.2–7.6 mg kg 1) were produced after
incubation at 30, 37 and 45 1C. However, all diacetyl
was reduced completely, probably to acetoin, after 2–8 h
incubation. Maximum acetoin levels were observed in
milk incubated with Lb. reuteri SD 2112 after 2, 4 and
12 h corresponding to incubation temperature of 45, 37
and 30 1C. In addition, Lb. rhamnosus GG (citrate
negative) produced 0.4–1.2 mg kg 1 diacetyl after 8–48 h
incubation at 20, 30, 37 and 45 1C, the highest amount at
20 1C after 48 h incubation. The diacetyl produced by
Lb. rhamnosus GG during the fermentation period was
not reduced, and this would undoubtedly make an
important contribution to the overall flavour of the milk
since the taste threshold of diacetyl is reported to be
around 0.03 mg kg 1 in homogenized milk (Reddy,
Lindsay, & Bills, 1969). Diacetyl is an important
compound determining the specific characteristics of
fermented milks (Oberman & Libudzisz, 1998). At very
low concentration (up to 5 mg kg 1) diacetyl is respon-
sible for the characteristic ‘‘buttery’’ aroma in milk
products (Oberman & Libudzisz, 1998). B. animalis
BB12, Lb. johnsonii LA1 and Lb. rhamnosus GG, all
citrate negative, produced 1.8–48.1 mg kg 1 acetoin
after 24–48 h incubation at the different temperatures
(Fig. 7). B. animalis BB12 produced the highest amount
of acetoin (48.1 mg kg 1) after 48 h incubation at 30 1C
and the lowest amount (1.8 mg kg 1) after 24 h incuba-
tion at 45 1C. It is possible that B. animalis BB12
produced some acetoin instead of acidic end-products to
maintain pH homeostasis since high levels of pyruvate
were reported after incubation at 30 1C for 18 h and the
pH was 4.1. Tsau (1992) reported that the conversion of
pyruvate to acetoin instead of acidic end-products
contributed to the maintenance of pH homeostasis in
Lb. plantarum. Lb. johnsonii LA1 and Lb. rhamnosus
GG produced 13.8 and 9.8 mg kg 1 after 48 h incubation
at 20 1C (results not shown).
Carbon dioxide can be produced via several metabolic
pathways, such as citrate breakdown (Marshall, 1987)
and by the pyruvate oxidase pathway (Hugenholtz,
1993). Carbon dioxide produced by the strains after 48 h
incubation varied from 68.4 to 4014 mg kg 1 after
incubation at the different temperatures (Fig. 8) and
was clearly connected with whether the individual strain
could degrade citrate and also which carbohydrate
fermentation pathway was used. The heterofermentative
and citrate-degrading Lb. reuteri SD 2112 produced the
highest amount of carbon dioxide, 3496–4014 mg kg 1,
and was little influenced by temperature. The homo-
fermentative and citrate-degrading Lb. acidophilus La5
and Lb. acidophilus 1748 produced 262–638 mg kg 1 and
527–753 mg kg 1, respectively, after 48 h incubation at
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996 H.M. Østlie et al. / International Dairy Journal 15 (2005) 989–997
Fig. 8. Production of carbon dioxide during fermentation at (a) 30 1C,
(b) 37 1C and (c) 45 1C with B. animalis BB12 (~), Lb. acidophilus La5
(&), Lb. acidophilus 1748 (K), Lb. reuteri SD2112 (m) and Lb.
johnsonii LA1 (W) in UHT milk supplemented with 0.5% (w/v)
tryptone and with Lb. rhamnosus GG (’) in UHT milk supplemented
with 0.75% (w/v) fructose. Vertical lines represent standard deviations.
the different temperatures, and the highest amounts
were produced after incubation at 45 1C and the lowest
after incubation at 30 1C. The homofermentative,
citrate-negative Lb. rhamnosus GG, Lb. johnsonii LA1
and B. animalis BB12 showed low CO2 production.
In the present work, growth and metabolism (in-
vestigated by quantification of key metabolites) were
followed for 48 h to investigate the survival of the
probiotic cells and their metabolites during fermentation
at different temperatures. The survival and viable cell
count of probiotic bacteria varied depending on the
strains and incubation conditions. However, the survival
of the probiotic strains in the fermented milk, except Lb.
reuteri SD 2112, was excellent during the 48 h fermenta-
tion. Five of the tested organisms showed viable cell
numbers between 6.5 and 9.6 log cfu mL 1 after 48 h
fermentation at 30, 37 and 45 1C. The various probiotic
strains tested in this study had very different metabolic
profiles in fortified milk with respect to known
organoleptically important compounds. The two Lb.
acidophilus strains were, however, most alike. These
differences in profiles would undoubtedly affect the
sensory quality of products made using these different
organisms. Our findings show the importance of
controlling the fermentation conditions, both time and
temperature, since the probiotic strains produce differ-
ent amount of metabolic products after a defined
fermentation time and temperature. Further studies will
address the metabolism of probiotic bacteria in milk as
affected by technological parameters such as heat
treatment of the milk and milk enrichment. The effect
of these different milk treatment regimes on the
metabolism, sensoric properties and survival of probio-
tic bacteria in the milk will be studied further.
Acknowledgements
The authors wish to acknowledge the expert technical
help of Kari Olsen for the GC and HPLC analyses. This
work was supported by a Grant from the Norwegian
Research council.
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